Alkylierende Substanzen – Bestimmung von Hämoglobin-Addukten als N-Methylvalin, N-Ethylvalin, N-Propylvalin, N-Butylvalin, N-(2-Hydroxypropyl)valin, N-(2-Hydroxyethyl)valin, N-(2-Cyanoethyl)valin, N-Benzylvalin und N-(2-Carbamoylethyl)valin in der Erythrozytenfraktion des Vollblutes mittels GC-MS/MS

Biomonitoring-Methode

Timo Christ¹ (Methodenentwicklung)
Dietmar Ellrich¹ (Methodenentwicklung)
Gabriele Leng¹ (Methodenentwicklung)
Christoph Schmidtkunz¹ (Methodenentwicklung)
Wolfgang Gries¹ (Methodenentwicklung)
Fabian Pilz² (Methodenprüfung)
Gerhard Scherer² (Methodenprüfung)
  Thomas Göen³ (Leitung der Arbeitsgruppe „Analysen in biologischem Material“ der Ständigen Senatskommission zur Prüfung gesundheitsschädlicher Arbeitsstoffe, Deutsche Forschungsgemeinschaft)
  Andrea Hartwig⁴ (Vorsitz der Ständigen Senatskommission zur Prüfung gesundheitsschädlicher Arbeitsstoffe, Deutsche Forschungsgemeinschaft)
  MAK Commission<sup>5

1</sup> Currenta GmbH & Co. OHG, CUR-SIT-SER-GS-BLM – Institut für Biomonitoring, Chempark Geb. Q 18, 51368 Leverkusen, Deutschland
² ABF – Analytisch-biologisches Forschungslabor GmbH, Semmelweisstraße 5, 82152 Planegg, Deutschland
³ Friedrich-Alexander-Universität Erlangen-Nürnberg, Institut und Poliklinik für Arbeits-, Sozial- und Umweltmedizin, Henkestraße 9–11, 91054 Erlangen, Deutschland
⁴ Institut für Angewandte Biowissenschaften, Abteilung Lebensmittelchemie und Toxikologie, Karlsruher Institut für Technologie (KIT), Adenauerring 20a, Geb. 50.41, 76131 Karlsruhe, Deutschland
⁵ Ständige Senatskommission zur Prüfung gesundheitsschädlicher Arbeitsstoffe, Deutsche Forschungsgemeinschaft, Kennedyallee 40, 53175 Bonn, Deutschland

Abstract

The working group “Analyses in Biological Materials” of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area developed and verified the presented biomonitoring method. Alkylating substances play an important role in many industrial processes and include n-alkyl and benzyl halides, α,β-unsaturated carbonyl compounds, epoxides, alkylnitrosamines, chloroalkyl ethers, dialkyl sulfates as well as alkyl alkane sulfonates. As alkylating substances are mainly used in industry, most exposure occurs in the workplace. The general population is primarily exposed through cigarette smoke, which contains a variety of alkylating substances. In addition, certain foods, especially highly heated, starchy foods, are a source of exposure to acrylamide, another alkylating substance. The aim of this work was to develop a selective method to simultaneously determine the adducts of different alkylating substances with the N-terminal valine of haemoglobin, namely N-methylvaline, N-ethylvaline, N-propylvaline, N-butylvaline, N-(2-hydroxypropyl)valine, N-(2-hydroxyethyl)valine, N-(2-cyanoethyl)valine, N-benzylvaline, and N-(2-carbamoylethyl)valine. The method has been comprehensively validated, and the reliability data have been confirmed by replication and verification of the procedure in a second, independent laboratory. For the determination of the adducts at the N-terminal valine of haemoglobin, erythrocytes are isolated from whole blood and subjected to lysis. Globin is precipitated from the haemoglobin solution and, after the addition of N-(2-ethoxyethyl)valine-alanine-anilide as internal standard, the alkylated N-terminal valines are derivatised and cleaved off by a modified Edman degradation using pentafluorophenyl isothiocyanate. The corresponding pentafluorophenyl thiohydantoin derivates are extracted with tert-butyl methyl ether, washed, and analysed by GC-MS/MS. The good precision and accuracy data show that the method is reliable and accurate. The method is both selective and sensitive, and the limits of detection between 10 and 70 pmol/g globin are sufficient to determine occupational exposure and, for some analytes, exposure of the general population to the respective alkylating substances.

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